Summary Lyme disease (LD) is the most commonly reported vector-borne disease in the United States with approximately 38,000 confirmed and probable cases in 2009. The currently recommended method for diagnosing LD is a two-tiered serology-based test that is limited in its ability to differentiate active from previous infection, diagnose early LD, and is not standardized among laboratories. These limitations can result in misinterpretations and have led to millions of diagnostic tests (FDA approved and non-approved) being performed each year for this infection. Thus, a fresh approach for improved diagnostic development is critical to the field. Studies applying the global evaluation of small molecule biomarkers, metabolomics, for the discovery of biosignatures and biomarkers for monitoring cancers and metabolic diseases have shown tremendous promise. Although this technology has not been widely applied for infectious diseases, it is recognized that infectious diseases are manifested based on alterations to the biochemistry of a biological system. Thus, as is now being shown with other infections, we hypothesize that metabolomics offers an innovative approach for identification of biosignatures and biomarkers of LD. This R21/R33 application proposes to, in the first two years, elucidate multiple metabolome-based biosignatures of LD; initiate identification of products that comprise these biosignatures; and establish tools for the elucidation of the Borrelia burgdorferi metabolome. Successful completion of these initial studies and tool development efforts will lead to efforts in Years 3-5 to qualify the LD biosignatures; complete the molecular identification of the biosignature products; and provide a comprehensive analysis of the B. burgdorferi metabolome. These activities encompassing the entirety of the R21/R33 program are framed within three Specific Aims: 1) Develop and qualify metabolic biosignatures that differentiate active LD from look-alike infections, and define the stages of disease including cured LD; 2) Identify and validate the chemical structures of the metabolites that comprise the biosignatures of LD; and 3) Evaluate the metabolic profile of B. burgdorferi. The overall goal at the end of five years will be to have qualified LD biosignatures that can be applied in clinical laboratories using mass spectrometry or another analytical technique suitable for a multi-analyte small molecule diagnostic.